title: TBK1 mediates critical effects of measles virus nucleocapsid protein (MVNP) on pagetic osteoclast formation.
abstract: Paget's disease of bone (PDB) is characterized by abnormal osteoclasts with unique characteristics that include increased sensitivity of osteoclast progenitors to 1,25(OH)2 D3 , receptor activator of NF-κB ligand (RANKL), and TNF-α; increased osteoclast numbers; and increased expression of IL-6 and several transcription factors. We recently reported that measles virus nucleocapsid protein (MVNP) plays a key role in the development of these abnormal osteoclasts. MVNP can induce the pagetic osteoclast phenotype in vitro and in vivo in TRAP-MVNP transgenic mice. However, the molecular mechanisms by which MVNP generates pagetic osteoclasts have not been determined. TANK-binding kinase 1 (TBK1) and IκB kinase-ϵ (IKKϵ) are IKK family members that complex with MVNP and activate both IRF3 and NF-κB pathways. MVNP increases the amount of TBK1 protein in bone marrow monocytes (BMM). Interestingly, we found that RANKL increased TBK1 and IKKϵ early in osteoclast differentiation, suggesting a possible role in normal osteoclastogenesis. However, only TBK1 is further increased in osteoclasts formed by TRAP-MVNP BMM owing to increased TBK1 protein stability. TBK1 overexpression induced IL6 promoter reporter activity, and elevated endogenous IL6 mRNA and p65 NF-κB, TAF12, and ATF7 proteins in several cell lines. Overexpression of TBK1 was insufficient to induce pagetic osteoclasts from WT BMM but synergized with MVNP to increase pagetic osteoclast formation from TRAP-MVNP BMM. BX795 inhibition of TBK1 impaired MVNP-induced IL-6 expression in both NIH3T3 cells and BMM, and shRNA knockdown of Tbk1 in NIH3T3 cells impaired IL-6 secretion induced by MVNP and decreased TAF12 and ATF7, factors involved in 1,25(OH)2 D3 hypersensitivity of pagetic osteoclasts. Similarly, Tbk1 knockdown in BMM from TRAP-MVNP and WT mice specifically impaired development of the MVNP-induced osteoclast pagetic phenotype. These results demonstrate that TBK1 plays a critical role in mediating the effects of MVNP on osteoclast differentiation and on the expression of IL-6, a key contributor to the pagetic osteoclast phenotype.
authors.affiliation: Department of Medicine/Hematology-Oncology, University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Quanhong
authors.initials: Q
authors.lastname: Sun
authors.forename: Bénédicte
authors.initials: B
authors.lastname: Sammut
authors.forename: Feng-Ming
authors.initials: FM
authors.lastname: Wang
authors.forename: Noriyoshi
authors.initials: N
authors.lastname: Kurihara
authors.forename: Jolene J
authors.initials: JJ
authors.lastname: Windle
authors.forename: G David
authors.initials: GD
authors.lastname: Roodman
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Galson
mesh_terms: D000135:Acid Phosphatase; D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism*; D015850:Interleukin-6 / Q000096:biosynthesis / Q000378:metabolism; D007527:Isoenzymes; D051379:Mice; D041681:NIH 3T3 Cells; D019590:Nucleocapsid Proteins / Q000378:metabolism*; D010001:Osteitis Deformans / Q000235:genetics / Q000517:prevention & control; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D053245:RANK Ligand / Q000494:pharmacology; D000071681:Tartrate-Resistant Acid Phosphatase; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D015850:Interleukin-6; D007527:Isoenzymes; D019590:Nucleocapsid Proteins; D053245:RANK Ligand; D014409:Tumor Necrosis Factor-alpha; C000590691:measles virus nucleocapsid protein; C498588:Tbk1 protein, mouse; D017346:Protein Serine-Threonine Kinases; D051550:I-kappa B Kinase; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase
keywords: IL-6; MVNP; OSTEOCLASTS; PAGET'S DISEASE; TBK1